Method of prognosing and diagnosing hereditary spastic paraplegia, mutant nucleic acid molecules and polypeptides

ABSTRACT

A method for diagnosing the presence of hereditary spastic paraplegia (HSP) or predicting the risk of developing HSP in a human subject, comprising detecting the presence or absence of a defect in a gene encoding a polypeptide comprising the sequence of FIG.  9  (SEQ ID NO: 19), in a nucleic acid sample of the subject, whereby the detection of the defect is indicative that the subject has or is at risk of developing HSP.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/858,354, filed on Nov. 13, 2006. The entire teachings of the aboveapplication are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to method of prognosing and diagnosinghereditary spastic paraplegia, mutant nucleic acid molecules andpolypeptides.

BACKGROUND OF THE INVENTION

Hereditary Spastic Paraplegia (HSP) has a worldwide prevalence between1-18 in 100,000¹⁻³ and is characterized by central motor system deficitsleading to lower limb spastic paraperesis.⁴⁻⁶ This is due to a “dyingback” phenomenon whereby upper motor neurons degenerate progressively,commencing with the longest axons.^(7,8) HSP can be classified into pureand complicated forms.⁶ In pure HSP, lower limb spasticity is the onlymajor presenting symptom. Alternatively, in complicated HSP, thisspasticity can be accompanied by other neurological or non-neurologicalsymptoms such as ataxia, dementia, mental retardation, deafness,epilepsy, ichthyosis, retinopathy, ocular neuropathy and extrapyramidaldisturbances.^(6,9) There is clinical heterogeneity within families,where age of onset and severity can differ markedly; between familiesthat map to the same locus; and certainly between families which map toseparate loci. This complicates genotype-phenotype correlations for HSP.

HSP is also extremely genetically heterogeneous. Eleven genes have beenidentified out of over 30 loci mapped (SPG1-33). This disease can betransmitted in a dominant (13 loci), a recessive (15 loci) or anX-linked manner (4 loci).⁹⁻¹¹ By far the most common locus for thedisease is SPG4, with mutations in the microtubule severing proteinspastin accounting for ˜40 percent of dominant HSP cases(MIM604277).^(12,13)

SPG8 is a pure form of hereditary spastic paraplegia with relativelylittle interfamilial variability in phenotype. SPG8 is considered to beone of the more aggressive subtypes of HSP with disease onset occurringfor patients as early as their 20 s or 30 s. It was first identified ina Caucasian family as a 6.2 cM region between the markers D8S1804 andD8S1774.¹⁴ The family contained 15 patients affected with spasticity,hyperreflexia, extensor plantar reflexes, lower limb weakness, decreasedvibration sensation and limited muscle wasting. The candidate region wasfurther reduced to 3.4 cM due to a lower recombinant in a second family,narrowing the interval between markers D8S1804 and D8S1179.¹⁵ Thisfamily as well as a third Brazilian family linked to SPG8 also presentedwith pure adult onset HSP.¹⁶ For two of the families, a muscle biopsywas performed;^(14,16) however, no gross histological or histochemicalabnormalities were observed. Ragged red fibers have been observed inmuscle biopsies of HSP patients with paraplegin mutations.¹⁷ These threefamilies thus present with relatively severe, pure spastic paraplegia.

HSP is one of the most genetically heterogeneous diseases, caused bymutations in at least 31 different genes. This means that >0.1% of genesin the human genome can be mutated resulting in one predominantneurological outcome: the degeneration of upper motor neuron axons. Thisheterogeneity may in part explain why it was originally difficult toidentify the eighth HSP locus, SPG8 leading to an expansion of thecandidate interval. The eighth HSP locus, SPG8, is on chromosome8p24.13. It is possible that two spastic paraplegia genes exist onchromosome 8q23-24, and the overlap of linkage results from both lociyielded a region between the two causative genes. This is similar to theSPG33 gene ZFVE27 which is in close proximity to the SPG9 (MIM 601162)and SPG27 (MIM609041) loci.²⁵ Alternatively, one originally reportedfamily may have had a false positive linkage result to chromosome 8.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

More specifically, in accordance with the present invention, there isprovided a method for diagnosing the presence of hereditary spasticparaplegia (HSP) or predicting the risk of developing HSP in a humansubject, comprising detecting the presence or absence of a defect in agene encoding a polypeptide comprising the sequence of FIG. 9 (SEQ IDNO: 19), in a nucleic acid sample of the subject, whereby the detectionof the defect is indicative that the subject has or is at risk ofdeveloping HSP.

In a specific embodiment of the method, said sample comprises DNA. Inanother specific embodiment, said sample comprises RNA. In anotherspecific embodiment, the defect is a missense or spice site mutation.

In another specific embodiment, the defect comprises a mutation in thegene resulting in a mutant polypeptide in which at least one amino acidresidue of FIG. 9 (SEQ ID NO: 19) is substituted with another amino acidresidue, and wherein the at least one amino acid residue is selectedfrom the group consisting of an asparagine residue at position 471, aleucine residue at position 619 and a valine residue at position 626. Inanother specific embodiment, the defect comprises a mutation in the generesulting in a mutant polypeptide in which amino acid residue 471 ofFIG. 9 (SEQ ID NO: 19) is substituted with an aspartate residue, or inwhich amino acid residue 619 of FIG. 9 (SEQ ID NO: 19) is substitutedwith a phenylalanine residue or in which amino acid residue 626 of FIG.9 (SEQ ID NO: 19) is substituted with a phenylalanine residue.

In another specific embodiment, the defect comprises a mutation in thegene, wherein the gene is as set forth in FIG. 8 (SEQ ID NO: 18), themutation being selected from the group consisting of a substitution of aguanine at position 2205 with another nucleotide, a substitution of aguanine at position 2186 with another nucleotide, and a substitution ofan adenine at position 1740 with another nucleotide. In another specificembodiment, the defect comprises a mutation in the gene, wherein thegene is as set forth in FIG. 8 (SEQ ID NO: 18), the mutation beingselected from the group consisting of a substitution of a guanine atposition 2205 with a thymine, a substitution of a guanine at position2186 with a cytosine, and a substitution of an adenine at position 1740with a guanine.

In accordance with another aspect of the present invention, there isprovided a method comprising the steps of: a) analyzing a nucleic acidtest sample containing the gene; b) comparing the results of saidanalysis of said sample of step a) with the results of an analysis of acontrol nucleic acid sample containing a wildtype strumpellin gene,wherein the wildtype strumpellin gene encodes a polypeptide comprisingthe sequence of FIG. 9 (SEQ ID NO: 19); and c) determining the presenceor absence of at least one defect in the strumpellin gene of the testsample.

In another specific embodiment of the method, the nucleic acid sample isamplified prior to analysis. In another specific embodiment, the defectis a mutation in the coding region of the strumpellin gene. In anotherspecific embodiment, the mutation is a missense or splice site mutation.

In another specific embodiment, the defect comprises a mutation in thegene resulting in a mutant polypeptide in which at least one amino acidresidue of FIG. 9 (SEQ ID NO: 19) is substituted with another amino acidresidue, and wherein the at least one amino acid residue is selectedfrom the group consisting of an asparagine residue at position 471, aleucine residue at position 619 and a valine residue at position 626. Inanother specific embodiment, the defect comprises a mutation in the generesulting in a mutant polypeptide in which amino acid residue 471 ofFIG. 9 (SEQ ID NO: 19) is substituted with an aspartate residue, or inwhich amino acid residue 619 of FIG. 9 (SEQ ID NO: 19) is substitutedwith a phenylalanine residue or in which amino acid residue 626 of FIG.9 (SEQ ID NO: 19) is substituted with a phenylalanine residue. Inanother specific embodiment, the defect comprises a mutation in thegene, the gene being as set forth in FIG. 8 (SEQ ID NO: 18), themutation being selected from the group consisting of a substitution of aguanine at position 2205 with another nucleotide, a substitution of aguanine at position 2186 with another nucleotide, and a substitution ofan adenine at position 1740 with another nucleotide. In another specificembodiment, the defect comprises a mutation in the gene, the gene beingas set forth in FIG. 8 (SEQ ID NO: 18), the mutation being selected fromthe group consisting of a substitution of a guanine at position 2205with a thymine, a substitution of a guanine at position 2186 with acytosine, and a substitution of an adenine at position 1740 with aguanine.

In another specific embodiment, the analysis is selected from the groupconsisting of: sequence analysis; fragment polymorphism assays;hybridization assays and computer based data analysis.

In accordance with another aspect of the present invention, there isprovided a method of detecting the presence or absence of a mutation ina strumpellin gene, said method comprising the steps of: a) analyzing anucleic acid test sample containing the gene; b) comparing the resultsof said analysis of said sample of step a) with the results of ananalysis of a control nucleic acid sample containing a wildtypestrumpellin gene, wherein the wildtype strumpellin gene comprises thesequence of FIG. 8 (SEQ ID NO: 18); and c) determining the presence orabsence of at least one defect in the strumpellin gene of the testsample.

In a specific embodiment, the nucleic acid sample is amplified prior toanalysis.

In another specific embodiment, the mutation comprises a mutation in thegene, the mutation being selected from the group consisting of asubstitution of a guanine at position 2205 with another nucleotide, asubstitution of a guanine at position 2186 with another nucleotide, anda substitution of an adenine at position 1740 with another nucleotide.In another specific embodiment, the mutation comprises a mutation in thegene, the mutation being selected from the group consisting of asubstitution of a guanine at position 2205 with a thymine, asubstitution of a guanine at position 2186 with a cytosine, and asubstitution of an adenine at position 1740 with a guanine.

In accordance with another aspect of the present invention, there isprovided a method of selecting a compound, said method comprising: (a)contacting a test compound with at least one biological systemdisplaying a defect in a gene encoding a polypeptide, the polypeptidecomprising the sequence of FIG. 9 (SEQ ID NO: 19), wherein the testcompound is selected if the polypeptide function, expression orconformation is modified in the presence of the test compound ascompared to that in the absence thereof.

In accordance with another aspect of the present invention, there isprovided a purified polypeptide comprising a sequence selected from thegroup consisting of the sequence in FIG. 11 (SEQ ID NO: 21), in FIG. 13(SEQ ID NO: 23), and in FIG. 15 (SEQ ID NO: 25).

In accordance with another aspect of the present invention, there isprovided a purified antibody that binds specifically to the polypeptideof the present invention.

In accordance with another aspect of the present invention, there isprovided a method of determining whether a biological sample containsthe polypeptide of the present invention, comprising contacting thesample with a purified ligand that specifically binds to thepolypeptide, and determining whether the ligand specifically binds tothe sample, the binding being an indication that the sample contains thepolypeptide.

In a specific embodiment, the ligand is a purified antibody.

In accordance with another aspect of the present invention, there isprovided an isolated nucleic acid molecule of no more than 300nucleotides comprising (a) a sequence of at least 19 contiguousnucleotides of the sequence of FIG. 10 (SEQ ID NO: 20), comprising thenucleotide at position 1740 of FIG. 10; (b) a sequence of at least 19contiguous nucleotides of the sequence of FIG. 12 (SEQ ID NO: 22),comprising the nucleotide at position 2186 of FIG. 12; (c) a sequence ofat least 19 contiguous nucleotides of the sequence of FIG. 14 (SEQ IDNO: 24), comprising the nucleotide at position 2205 of FIG. 14; (d) thecomplement of the sequence in (a), (b) or (c); or (e) a sequence of atleast 19 contiguous nucleotides hybridizable under high stringencyconditions to the sequence in (a), (b), (c) or (d).

In accordance with another aspect of the present invention, there isprovided a vector comprising the nucleic acid molecule of the presentinvention. In accordance with another aspect of the present invention,there is provided a recombinant host cell comprising the vector of thepresent invention.

In accordance with another aspect of the present invention, there isprovided an array of nucleic acid molecules attached to a solid support,the array comprising an oligonucleotide hybridizable to one of thenucleic acid molecules of the present invention.

In accordance with another aspect of the present invention, there isprovided an isolated nucleic acid molecule comprising the sequence of(a) FIG. 10 (SEQ ID NO: 20); (b) FIG. 12 (SEQ ID NO: 22); (c) FIG. 14(SEQ ID NO: 24); or (d) the complement of the sequence in (a), (b) or(c). In accordance with another aspect of the present invention, thereis provided a vector comprising the nucleic acid molecule of the presentinvention. In accordance with another aspect of the present invention,there is provided a recombinant host cell comprising the vector of thepresent invention.

In accordance with another aspect of the present invention, there isprovided an isolated nucleic acid molecule encoding a polypeptidecomprising the amino acid sequence of (a) FIG. 11 (SEQ ID NO: 21); (b)FIG. 13 (SEQ ID NO: 23); (c) FIG. 15 (SEQ ID NO: 25); or (d) thecomplement of the sequence in (a), (b) or (c). In accordance withanother aspect of the present invention, there is provided a vectorcomprising the nucleic acid molecule of the present invention. Inaccordance with another aspect of the present invention, there isprovided a recombinant host cell comprising the vector of the presentinvention.

In specific embodiments of the methods of the present invention, thesubject is pre-diagnosed as being a likely candidate for developing HSP.

In accordance with another aspect of the present invention, there isprovided a purified polypeptide consisting of a sequence selected fromthe group consisting of the sequence in FIG. 11 (SEQ ID NO: 21), in FIG.13 (SEQ ID NO: 23), and in FIG. 15 (SEQ ID NO: 25).

In accordance with another aspect of the present invention, there isprovided a method of stratifying a subject having hereditary spasticparaplegia (HSP) comprising: detecting a defect in a strumpellin proteinactivity and/or in a nucleic acid encoding the protein in a biologicalsample; whereby the results of the detecting step enables thestratification of the subject having HSP as belonging to a HSP subclass.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawings will be provided by the Office upon request and payment of thenecessary fee.

FIG. 1 are pedigrees for families with KIAA0196 mutations. A) FamilyFSP24, B) Family FSP29, C) Family FSP34, D) Family FSP91. Blackenedboxes represent affected individuals, and a diagonal line through thesymbol means the individual is deceased. A vertical black bar indicatesan individual with an unconfirmed phenotype. Sex of each individual hasbeen masked to preserve confidentiality. Individuals marked “P”represent proximal recombinants and “D” is the distal recombinant. Astar indicates that DNA and clinical information have been collected forthe particular individual. The age of onset of affected individuals islisted below each symbol, although this information is not available foreach patient. All collected affected patients are heterozygous for a c.2205C->T mutation (pedigrees A, B and C) or a c.A1740G mutation(pedigree D) in KIAA0196 (NCBI accession # NM_(—)014846.3);

FIG. 2 is a region spanning the SPG8 locus. A) Markers defining theborders of each described SPG8 family and their scaled position onchromosome 8q24.13. B) Candidate region used to search for theSPG8-located gene between markers D8S1804 and D8S1774. Genes in theregion are shown in their observed orientation. C) The 28-exon KIAA0196gene drawn to scale with the location of 3 mutations in exons 11, 14 and15 highlighted;

FIG. 3 presents a mutation analysis of the KIAA0196 gene. A-C) Sequencetrace of an HSP patient above the sequence trace of a controlindividual. Exon 15 (A), 14 (B) and 11 (C) heterozygous point mutationsare indicated. D) Multiple sequence alignment for strumpellin homologuessurrounding the two coding changes (boxed) (human (SEQ ID NO: 7);orangutan (SEQ ID NO: 8); rat (SEQ ID NO: 9); mouse (SEQ ID NO: 10); dog(SEQ ID NO: 11); chicken (SEQ ID NO: 12); zebrafish (SEQ ID NO: 13);fruit fly (SEQ ID NO: 14); C. elegans (SEQ ID NO: 15); frog (SEQ ID NO:16); and amoeba (SEQ ID NO: 17). The Probcons™ (v.1.09) program³⁸ wasused for cluster analysis. E) RT-PCR of multiple brain regions using aKIAA0196-specific probe. F) Northern blot of the KIAA0196 transcriptusing 30 ug of total RNA and a 1 kb C-terminal probe;

FIG. 4 presents a three-dimensional modelling of strumpellin usingd1dn1b as a template using SwissProt™ database viewer. Two helices fromthe 1159aa protein are shown including amino acids 614-634 in one alphahelix and amino acids 662-672 from a nearby alpha helix in theantiparallel direction. A) Residues L619 and V626 are in the sameorientation in an alpha helix opposite a second helix in an antiparalleldirection. Only residue side-chains which are closest in physical spaceare shown. B) The L619F mutation adds a bulky phenylalanine side-groupwhich likely exceeds the space available between the two alpha helicesC) The V626F mutation. The epsilon carbon of the F626 aromatic ring alsomay force apart the two alpha-helices and impinges on Q666;

FIG. 5 shows the results of a zebrafish knockdown and rescue of KIAA0196function. A) The gross morphological features of wildtype zebrafish aredepicted at 3 days post fertilization (dpf). B) Injection of a 5base-pair mismatch morpholino results in no obvious phenotype. C, D) TheKIAAMO injected fish present with a severely curly tail (C) or with aslightly curly tail (D). Their heart cavities are also enlarged, whichis commonly seen in injected fish. E, F) When the KIAAMO is injectedalong with normal human KIAA0196 mRNA, the fish partially develop thecurly tail (F) or not at all (E) depending on the injected quantity G),H) The phenotype is not alleviated when the KIAAMO is injected with themutant forms of the human mRNA. These fishes resemble the KIAAMO fish;

FIG. 6 presents an immunohistochemical analysis in zebrafish of theKIAA0196 knockdown phenotype. A) The motor neurons in the ventral rootsof zebrafish are segmented and oriented at 3 dpf. The spinal cordconsists of the cell bodies of motor neurons and interneuron bundles.The picture was taken near the gut of the fish. B) The mismatch controlhas a similar motor neuron distribution compared to the wildtype. C, E,F) Zebrafish injected with KIAAMO and fish co-injected with mutant mRNAhave shorter, branching motor neurons which are not oriented. D)Wildtype co-injections partially rescue the motor neuron phenotype; theaxons are longer and oriented;

FIG. 7 presents a western blot of the KIAA0196 protein. A) A westernblot was prepared using whole cell lysates of HeLa cells transientlyexpressing the different pCS2-KIAA0196 vectors that incorporate amyc-tag (WT, x14 and x15). The PVDF membrane was immunodetected using anantibody (9E10, Invitrogen) directed against the protein myc-tagportion. The KIAA196 protein migrates to its predicted MW (about 134kDa). The ctl lane corresponds to the untransfected cells. B) Apolyclonal antibody was generated against amino acids 652-669(VPTRLDKDKLRDYAQLGP (SEQ ID NO: 37)) of the KIAA0196 protein. Thisantibody was able to recognize the wildtype and L619F KIAA0196 proteinsequence;

FIG. 8 is the human wildtype KIAA0196 nucleotide sequence(NM_(—)014846.3) (SEQ ID NO: 18);

FIG. 9 is the human wildtype KIAA0196 amino acid sequence (NP_(—)055661)(SEQ ID NO: 19);

FIG. 10 is the mutated human KIAA0196 N471D (c.1740a>g) nucleotidesequence (SEQ ID NO: 20);

FIG. 11 is the mutated human KIAA0196 N471D amino acid sequence(NP_(—)055661) (SEQ ID NO: 21);

FIG. 12 is the mutated human KIAA0196 L619F (c.2186g>c) nucleotidesequence (SEQ ID NO: 22);

FIG. 13 is the mutated human KIAA0196 L619F amino acid sequence(NP_(—)055661) (SEQ ID NO: 23);

FIG. 14 is the mutated human KIAA0196 V626F (c.2205g>t) nucleotidesequence (SEQ ID NO: 24);

FIG. 15 is the mutated human KIAA0196 V626F amino acid sequence(NP_(—)055661) (SEQ ID NO: 25); and

FIG. 16 is a multi-species alignment of the human KIAA0196 (strumpellin)amino acid sequence with other species equivalents: Homo sapiens(Q12768) (SEQ ID NO: 26), Canis familiaris (XP_(—)532327) (SEQ ID NO:30), Pan troglodytes (XP_(—)519952) (SEQ ID NO: 27), Drosophilamelanogastar (CG12272) (SEQ ID NO: 33), Caenorhabditis elegans (CE13235)(SEQ ID NO: 35), Xenopus tropicalis (MGC89323) (SEQ ID NO: 32), Rattusnorvegicus (XP_(—)343250) (SEQ ID NO: 29); Danio rerio (BC045490) (SEQID NO: 34), Gallus gallus (XP_(—)418441) (SEQ ID NO: 31), Dictyosteliumdiscoideum (EAL63144) (SEQ ID NO: 36), and Mus musculus (NP_(—)705776.2)(SEQ ID NO: 28). The ruler above the alignment corresponds to the aminoacid position of the human KIAA0196 protein (NP_(—)055661).

DETAILED DESCRIPTION OF THE INVENTION

Encompassed by the present invention are methods of diagnosingSPG8-associated hereditary spastic paraplegia, or predicting the risk ofHSP by detecting mutations associated with the SPG8 locus. TheApplicants identified four families linked to the SPG8 locus. Genes werescreened in an expanded candidate SPG8 locus defined by these fourfamilies along with the British and Brazilian family describedpreviously.^(15,16) This led to the identification of three pointmutations in the KIAA0196 gene encoding the strumpellin protein productin these six families. One mutation, V626F, segregated in four largeNorth American families with European ancestry. An L619F mutation wasfound in the Brazilian family. The third mutation, N471D, was identifiedin a smaller family of European origin, and lies in a spectrin domain.None of these mutations were identified in 500 control individuals. Boththe L619 and V626 residues are strictly conserved across species andlikely have a notable effect on the structure of the protein product,strumpellin. Rescue studies with human mRNA injected in zebrafishtreated with morpholino oligonucleotides to knockdown the endogenousprotein showed that mutations at these two residues impaired the normalfunction of the KIAA0196 gene. Recovery of a normal strumpellin activitynevertheless resulted in recovering normal muscle function. To theApplicant knowledge, there is no other gene than the KIAA0196 geneinvolved in the SPG8-associated hereditary spastic paraplegia.

DEFINITIONS

As used herein the expressions “risk of developing HSP” or “likelycandidate for developing HSP” include subjects suspected of having HSPor subjects of which a least one parent has HSP.

As used herein the terms “defect”, “alteration” or “variation” refers toa mutation or polymorphism in the KIAA0196 gene (also referred to hereinas the strumpellin gene) that affects the function, expression(transcription or translation) or conformation of the protein(strumpellin) that it encodes. Mutations encompassed by the presentinvention can be any mutation the KIAA0196 gene that results in thedisruption of the function, expression or conformation of the encodedprotein, including the complete absence of expression of the encodedprotein and can include, for example, missense and nonsense mutations,insertions and deletions. Without being so limited, mutationsencompassed by the present invention may alter splicing the mRNA (splicesite mutation) or cause a shift in the reading frame (frameshift).Without being so limited, modifications of the function of strumpellincan be observed with methods such as the zebrafish knockouts experimentspresented in Example 6 below.

Also encompassed by the present invention are methods of detecting novelmutations of interest in the strumpellin gene that are associated withHSP. A mutation of interest is any mutation detected in a gene sampleobtained from a human subject, having, or suspected of having, HSP. Forexample, the nucleic acid sequence of a strumpellin gene obtained from ahuman subject can be compared with the nucleic acid sequence of a wildtype (control) strumpellin gene and differences in the nucleotidesequence determined. A difference in the nucleotide sequence of the genefrom the human subject is indicative of a mutation associated with HSP.Modifications of a protein encoded by the “different” human gene can beanalyzed by various methods, for example, in the zebra fish assaydescribed herein, to evaluate expression or function of the encodedprotein. Further, the familial history of HSP, or present symptoms ofthe human subject can be reviewed, and a determination of theassociation of the novel mutation with HSP can be made. Thus, additionalmutations in the strumpellin gene can be associated with, and diagnosticof, HSP.

The articles “a,” “an” and “the” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle.

The term “including” and “comprising” are used herein to mean, and reused interchangeably with, the phrases “including but not limited to”and “comprising but not limited to”.

The terms “such as” are used herein to mean, and is used interchangeablywith, the phrase “such as but not limited to”.

As used herein the term “subject” is meant to refer to any mammalincluding human, mouse, rat, dog, cat, pig, monkey, horse, etc. In aparticular embodiment, it refers to a human.

As used herein the term a suitable “control nucleic acid sample” ismeant to refer to a nucleic acid sample (RNA, DNA) that does not comefrom a subject known to suffer from HSP (control subject). For example,the control can be a wild type strumpellin gene which does not contain avariation in its nucleic acid sequence. Also, as used herein, a suitablecontrol can be a fragment or portion of the wilt type gene that does notinclude the defect/variation that is the mutation of interest (that is,the mutation to be detected in an assay).

As used herein the terms “subject nucleic acid sample” are meant torefer to any biological sample from the subject from whom nucleic acidsample (RNA, DNA) can be extracted, namely any subject tissue or celltype including saliva and blood.

The present invention also relates to methods for the determination ofthe level of expression of transcripts or translation product of asingle gene such as KIAA0196. The present invention thereforeencompasses any known method for such determination including real timePCR and competitive PCR, Northern blots, nuclease protection, plaquehybridization and slot blots. For example, assays commonly used toanalyze nucleic acid polymorphisms can include sequencing all, or aportion of, the nucleic acid to detect a variation in the nucleotidesequence. Such assays can include fragment polymorphism analysis,nucleic acid hybridization assays and computerized nucleotide or aminoacid sequence comparisons

The present invention also concerns isolated nucleic acid moleculesincluding probes. In specific embodiments, the isolated nucleic acidmolecules have no more than 300, or no more than 200, or no more than100, or no more than 90, or no more than 80, or no more than 70, or nomore than 60, or no more than 50, or no more than 40 or no more than 30nucleotides. In specific embodiments, the isolated nucleic acidmolecules have at least 20, or at least 30, or at least 40 nucleotides.In other specific embodiments, the isolated nucleic acid molecules haveat least 20 and no more than 300 nucleotides. In other specificembodiments, the isolated nucleic acid molecules have at least 20 and nomore than 200 nucleotides. In other specific embodiments, the isolatednucleic acid molecules have at least 20 and no more than 100nucleotides. In other specific embodiments, the isolated nucleic acidmolecules have at least 20 and no more than 90 nucleotides. In otherspecific embodiments, the isolated nucleic acid molecules have at least20 and no more than 80 nucleotides. In other specific embodiments, theisolated nucleic acid molecules have at least 20 and no more than 70nucleotides. In other specific embodiments, the isolated nucleic acidmolecules have at least 20 and no more than 60 nucleotides. In otherspecific embodiments, the isolated nucleic acid molecules have at least20 and no more than 50 nucleotides. In other specific embodiments, theisolated nucleic acid molecules have at least 20 and no more than 40nucleotides. In other specific embodiments, the isolated nucleic acidmolecules have at least 20 and no more than 30 nucleotides. In otherspecific embodiments, the isolated nucleic acid molecules have at least30 and no more than 300 nucleotides. In other specific embodiments, theisolated nucleic acid molecules have at least 30 and no more than 200nucleotides. In other specific embodiments, the isolated nucleic acidmolecules have at least 30 and no more than 100 nucleotides. In otherspecific embodiments, the isolated nucleic acid molecules have at least30 and no more than 90 nucleotides. In other specific embodiments, theisolated nucleic acid molecules have at least 30 and no more than 80nucleotides. In other specific embodiments, the isolated nucleic acidmolecules have at least 30 and no more than 70 nucleotides. In otherspecific embodiments, the isolated nucleic acid molecules have at least30 and no more than 60 nucleotides. In other specific embodiments, theisolated nucleic acid molecules have at least 30 and no more than 50nucleotides. In other specific embodiments, the isolated nucleic acidmolecules have at least 30 and no more than 40 nucleotides.

Probes of the invention can be utilized with naturally occurringsugar-phosphate backbones as well as modified backbones includingphosphorothioates, dithionates, alkyl phosphonates and α-nucleotides andthe like. Modified sugar-phosphate backbones are generally known. Probesof the invention can be constructed of either ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA), and preferably of DNA.

The types of detection methods in which probes can be used includeSouthern blots (DNA detection), dot or slot blots (DNA, RNA), andNorthern blots (RNA detection). Although less preferred, labeledproteins could also be used to detect a particular nucleic acid sequenceto which it binds. Other detection methods include kits containingprobes on a dipstick setup and the like.

As used herein the terms “detectably labeled” refer to a marking of aprobe in accordance with the presence invention that will allow thedetection of the mutation of the present invention. Although the presentinvention is not specifically dependent on the use of a label for thedetection of a particular nucleic acid sequence, such a label might bebeneficial, by increasing the sensitivity of the detection. Furthermore,it enables automation. Probes can be labeled according to numerous wellknown methods. Non-limiting examples of labels include 3H, 14C, 32P, and35S, Non-limiting examples of detectable markers include ligands,fluorophores, chemiluminescent agents, enzymes, and antibodies. Otherdetectable markers for use with probes, which can enable an increase insensitivity of the method of the invention, include biotin andradionucleotides. It will become evident to the person of ordinary skillthat the choice of a particular label dictates the manner in which it isbound to the probe.

As commonly known, radioactive nucleotides can be incorporated intoprobes of the invention by several methods. Non-limiting examplesthereof include kinasing the 5′ ends of the probes using gamma 32P ATPand polynucleotide kinase, using the Klenow fragment of Pol I of E. coliin the presence of radioactive dNTP (e.g. uniformly labeled DNA probeusing random oligonucleotide primers in low-melt gels), using the SP6/T7system to transcribe a DNA segment in the presence of one or moreradioactive NTP, and the like.

The present invention also relates to methods of selecting compounds. Asused herein the term “compound” is meant to encompass natural, syntheticor semi-synthetic compounds, including without being so limitedchemicals, macromolecules, cell or tissue extracts (from plants oranimals), nucleic acid molecules, peptides, antibodies and proteins.

The present invention also relates to arrays. As used herein, an “array”is an intentionally created collection of molecules which can beprepared either synthetically or biosynthetically. The molecules in thearray can be identical or different from each other. The array canassume a variety of formats, e.g., libraries of soluble molecules;libraries of compounds tethered to resin beads, silica chips, or othersolid supports.

As used herein “array of nucleic acid molecules” is an intentionallycreated collection of nucleic acids which can be prepared eithersynthetically or biosynthetically in a variety of different formats(e.g., libraries of soluble molecules; and libraries of oligonucleotidestethered to resin beads, silica chips, or other solid supports).Additionally, the term “array” is meant to include those libraries ofnucleic acids which can be prepared by spotting nucleic acids ofessentially any length (e.g., from 1 to about 1000 nucleotide monomersin length) onto a substrate. The term “nucleic acid” as used hereinrefers to a polymeric form of nucleotides of any length, eitherribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs),that comprise purine and pyrimidine bases, or other natural, chemicallyor biochemically modified, non-natural, or derivatized nucleotide bases.The backbone of the polynucleotide can comprise sugars and phosphategroups, as may typically be found in RNA or DNA, or modified orsubstituted sugar or phosphate groups. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. Thus the terms nucleoside, nucleotide,deoxynucleoside and deoxynucleotide generally include analogs such asthose described herein. These analogs are those molecules having somestructural features in common with a naturally occurring nucleoside ornucleotide such that when incorporated into a nucleic acid oroligonucleotide sequence, they allow hybridization with a naturallyoccurring nucleic acid sequence in solution. Typically, these analogsare derived from naturally occurring nucleosides and nucleotides byreplacing and/or modifying the base, the ribose or the phosphodiestermoiety. The changes can be tailor made to stabilize or destabilizehybrid formation or enhance the specificity of hybridization with acomplementary nucleic acid sequence as desired.

As used herein “solid support”, “support”, and “substrate” are usedinterchangeably and refer to a material or group of materials having arigid or semi-rigid surface or surfaces. In many embodiments, at leastone surface of the solid support will be substantially flat, although insome embodiments it may be desirable to physically separate synthesisregions for different compounds with, for example, wells, raisedregions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations.

Any known nucleic acid arrays can be used in accordance with the presentinvention. For instance, such arrays include those based on short orlonger oligonucleotide probes as well as cDNAs or polymerase chainreaction (PCR) products. Other methods include serial analysis of geneexpression (SAGE), differential display, as well as subtractivehybridization methods, differential screening (DS), RNA arbitrarilyprimer (RAP)-PCR, restriction endonucleolytic analysis of differentiallyexpressed sequences (READS), amplified restriction fragment-lengthpolymorphisms (AFLP).

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridization are sequence dependent, andare different under different environmental parameters. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, 1984; T_(m) 81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, andL is the length of the hybrid in base pairs. T_(m) is reduced by about1° C. for each 1% of mismatching; thus, T_(m), hybridization, and/orwash conditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point I forthe specific sequence and its complement at a defined ionic strength andpH. However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point I;moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the thermal melting point I; lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point I. Using theequation, hybridization and wash compositions, and desired T, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point T_(m)for the specific sequence at a defined ionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see 64 for a description of SSCbuffer). Often, a high stringency wash is preceded by a low stringencywash to remove background probe signal. An example medium stringencywash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45°C. for 15 minutes. An example low stringency wash for a duplex of, e.g.,more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. Forshort probes (e.g., about 10 to 50 nucleotides), stringent conditionstypically involve salt concentrations of less than about 1.5 M, morepreferably about 0.01 to 1.0 M, Na ion concentration (or other salts) atpH 7.0 to 8.3, and the temperature is typically at least about 30° C.and at least about 60° C. for long robes (e.g., >50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

Washing with a solution containing tetramethylammonium chloride (TeMAC)could allow the detection of a single mismatch using oligonucleotidehybridization since such mismatch could generate a 10° C. difference inthe annealing temperature. The formulation to determine the washingtemperature is Tm (° C.)=]−682 (L⁻¹)+97 where L represents the length ofthe oligonucleotide that will be used for the hybridization. Inprinciple, a single mismatch will generate a 10° C. drop in theannealing so that a temperature of 57° C. should only detect mutantsharbouring the T mutation.

The present invention relates to a kit for diagnosing HSP and/orpredicting whether a subject is at risk of developing HSP comprising anisolated nucleic acid, a protein or a ligand such as an antibody inaccordance with the present invention. For example, a compartmentalizedkit in accordance with the present invention includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers or strips of plastic orpaper. Such containers allow the efficient transfer of reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated and the agents or solutions of each containercan be added in a quantitative fashion from one compartment to another.Such containers will include a container which will accept the subjectsample (DNA genomic nucleic acid, cell sample or blood samples), acontainer which contains in some kits of the present invention, theprobes used in the methods of the present invention, containers whichcontain enzymes, containers which contain wash reagents, and containerswhich contain the reagents used to detect the extension products. Kitsof the present invention may also contain instructions to use theseprobes and or antibodies to diagnose HSP or predict whether a subject isat risk of developing HSP.

As used herein the terminology “biological sample” refers to any solidor liquid sample isolated from a subject. In a particular embodiment, itrefers to any solid or liquid sample isolated from a human subject.Without being so limited it includes a biopsy material, blood, saliva,synovial fluid, urine, amniotic fluid and cerebrospinal fluid.

As used herein the terminology “biological system” is a cell, a tissue,an organ or an organism. Without being so limited, organisms include azebrafish.

As used herein the terminology “blood sample” is meant to refer toblood, plasma or serum.

As used herein the term “purified” in the expression “purifiedpolypeptide” means altered “by the hand of man” from its natural state(i.e. if it occurs in nature, it has been changed or removed from itsoriginal environment) or it has been synthesized in a non-naturalenvironment (e.g., artificially synthesized). These terms do not requireabsolute purity (such as a homogeneous preparation) but insteadrepresents an indication that it is relatively more pure than in thenatural environment. For example, a protein/peptide naturally present ina living organism is not “purified”, but the same protein separated(about 90-95% pure at least) from the coexisting materials of itsnatural state is “purified” as this term is employed herein.

Similarly, as used herein, the term “purified” in the expression“purified antibody” is simply meant to distinguish man-made antibodyfrom an antibody that may naturally be produced by an animal against itsown antigens. Hence, raw serum and hybridoma culture medium containinganti-strumpellin antibody are “purified antibodies” within the meaningof the present invention.

As used herein, the term “ligand” broadly refers to natural, syntheticor semi-synthetic molecules. The term “molecule” therefore denotes forexample chemicals, macromolecules, cell or tissue extracts (from plantsor animals) and the like. Non limiting examples of molecules includenucleic acid molecules, peptides, antibodies, carbohydrates andpharmaceutical agents. The ligand appropriate for the present inventioncan be selected and screened by a variety of means including randomscreening, rational selection and by rational design using for exampleprotein or ligand modeling methods such as computer modeling. The terms“rationally selected” or “rationally designed” are meant to definecompounds which have been chosen based on the configuration ofinteracting domains of the present invention. As will be understood bythe person of ordinary skill, macromolecules having non-naturallyoccurring modifications are also within the scope of the term “ligand”.For example, peptidomimetics, well known in the pharmaceutical industryand generally referred to as peptide analogs can be generated bymodeling as mentioned above.

Antibodies

As used herein, the term “anti-strumpellin antibody” or “immunologicallyspecific anti-strumpellin antibody” refers to an antibody thatspecifically binds to (interacts with) a strumpellin protein anddisplays no substantial binding to other naturally occurring proteinsother than the ones sharing the same antigenic determinants as thestrumpellin protein. The term antibody or immunoglobulin is used in thebroadest sense, and covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies,and antibody fragments so long as they exhibit the desired biologicalactivity. Antibody fragments comprise a portion of a full lengthantibody, generally an antigen binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments, diabodies, linear antibodies, single-chain antibodymolecules, single domain antibodies (e.g., from camelids), shark NARsingle domain antibodies, and multispecific antibodies formed fromantibody fragments. Antibody fragments can also refer to bindingmoieties comprising CDRs or antigen binding domains including, but notlimited to, VH regions (V_(H), V_(H)-V_(H)), anticalins, PepBodies™,antibody-T-cell epitope fusions (Troybodies) or Peptibodies.Additionally, any secondary antibodies, either monoclonal or polyclonal,directed to the first antibodies would also be included within the scopeof this invention.

In general, techniques for preparing antibodies (including monoclonalantibodies and hybridomas) and for detecting antigens using antibodiesare well known in the art (Campbell, 1984, In “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology”, Elsevier Science Publisher, Amsterdam, The Netherlands) and inHarlow et al., 1988 (in: Antibody A Laboratory Manual, CSHLaboratories). The term antibody encompasses herein polyclonal,monoclonal antibodies and antibody variants such as single-chainantibodies, humanized antibodies, chimeric antibodies andimmunologically active fragments of antibodies (e.g. Fab and Fab′fragments) which inhibit or neutralize their respective interactiondomains in Hyphen and/or are specific thereto.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc), intravenous (iv) or intraperitoneal (ip) injectionsof the relevant antigen with or without an adjuvant. It may be useful toconjugate the relevant antigen to a protein that is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example, maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals may be immunized against the antigen, immunogenic conjugates, orderivatives by combining the antigen or conjugate (e.g., 100 μg forrabbits or 5 μg for mice) with 3 volumes of Freund's complete adjuvantand injecting the solution intradermally at multiple sites. One monthlater the animals are boosted with the antigen or conjugate (e.g., with⅕ to 1/10 of the original amount used to immunize) in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Preferably, for conjugateimmunizations, the animal is boosted with the conjugate of the sameantigen, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (e.g., U.S. Pat. No. 6,204,023). Monoclonalantibodies may also be made using the techniques described in U.S. Pat.Nos. 6,025,155 and 6,077,677 as well as U.S. Patent ApplicationPublication Nos. 2002/0160970 and 2003/0083293 (see also, e.g.,Lindenbaum et al., 2004).

In the hybridoma method, a mouse or other appropriate host animal, suchas a rat, hamster or monkey, is immunized (e.g., as hereinabovedescribed) to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the antigen used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (see, e.g.,Goding 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

This invention will be described herein below, referring to specificembodiments and figures, the purpose of which is to illustrate theinvention rather than to limit its scope.

Example 1 Material and Methods

Subjects

Protocols were approved by the ethics committee of the Centrehospitalier de l'Université de Montréal (CHUM). Patients gave informedconsent after which patient information and blood was collected. DNA wasextracted from peripheral blood using standard protocols.

Genotyping and Locus Exclusion

PCR amplified fragments incorporating α-35S-dATP were resolved on 6%denaturing polyacrylamide gels. Alleles were run alongside an M13mp18sequence ladder and scored based on allele sizes and frequencies fromthe Fondation Jean Dausset CEPH database (http://www.cephb.fr/). LODscore calculations and multipoint analysis were performed using theMLINK program of the LINKMAP software package.¹⁸

Mutation Screening

The 28 exons of KIAA0196 were screened by automated sequencing,including at least 50 bp of each intronic region. Primers were designedusing the PrimerSelect™ program (Lasergene) and were synthesized byInvitrogen Canada Inc. Primer sequences and amplification conditions foreach exon are listed in Table 1 below.

TABLE 1 Primers and conditions for KIAA0196 KIAA0196x1FGCCAAGAGTGTTAATCTAGCAAAGTC (SEQ ID NO: 38) KIAA0196x1RTTCATGGTTCCCAGAGAAAACACG (SEQ ID NO: 39) KIAA0196x2FTCTGCTTTAAGTTTGGGATGTCTA (SEQ ID NO: 40) KIAA0196x2RTTAAGATGACCAGTGCCACAGGTA (SEQ ID NO: 41) KIAA0196x3FAATATCAAACTGTGGCCCTAAATC (SEQ ID NO: 42) KIAA0196x3RTACACCGAGGAGGCTCATAACTTC (SEQ ID NO: 43) KIAA0196x4FCATCCCAGCCATCTGTCCTGATAC (SEQ ID NO: 44) KIAA0196x4RACATACACTGCATTTTACCGACAGC (SEQ ID NO: 45) KIAA0196x5FAATGGAATTCTACTTTATTGGACT (SEQ ID NO: 46) KIAA0196x5RCTCAAAAGGTTTTAAAAGGTTCTACC (SEQ ID NO: 47) KIAA0196x6FTGGGCTTTGGAAAAACTGATGTGTCT (SEQ ID NO: 48) KIAA0196x6RAAGTTTACCTAAGTGATGTTATGTCC (SEQ ID NO: 49) KIAA0196x7FCAAAAAGCAACGTTAATAGGTGTAA (SEQ ID NO: 50) KIAA0196x7RATCATTGCATTAAATTATCTAAGTG (SEQ ID NO: 51) KIAA0196x8FTTAATCACAGCCAGAACTAGGATGTAG (SEQ ID NO: 52) KIAA0196x8RGACAGGGGAGAGCTTTTCAGGTATGCT (SEQ ID NO: 53) KIAA0196x9FTGGCACTCCATGTCAGATTCAACTGT (SEQ ID NO: 54) KIAA0196x9RATGTCTATATTCCCCATTAGG (SEQ ID NO: 55) KIAA0196x10FCAGGGTCAATGTTAATTTATAGTGTA (SEQ ID NO: 56) KIAA0196x10RAGATGGAGGCCAACTGTGACTCTC (SEQ ID NO: 57) KIAA0196x11FTGCTCCAGGCATTTTTGTCG (SEQ ID NO: 58) KIAA0196x11RGAACAGACTGCTGGGTGGGTCATA (SEQ ID NO: 59) KIAA0196x12ATGAGCACCATAGAGTCCATTCAGG (SEQ ID NO: 60) and 13F KIAA0196x12ATTATGCTCTCGTGGAAAAACTGCTA (SEQ ID NO: 61) and 13R KIAA0196x14FCTTTTTGAAACAAGAAACAGATATACC (SEQ ID NO: 62) KIAA0196x14RGGCAAGTAAAAACATCTGTACATCCAC (SEQ ID NO: 63) KIAA0196x15FTTTGCAGCATTTTTAGAAGGATTAGC (SEQ ID NO: 64) KIAA0196x15RTTCCCCTGAGAATACTGAGGCGAACA (SEQ ID NO: 65) KIAA0196x16FGGAGGCCAGGGAAGGGGAGGTTACC (SEQ ID NO: 66) KIAA0196x16RGGAATGTCAAACAGCCAGATGATGT (SEQ ID NO: 67) KIAA0196x17FACTTTGCTGAAATAAACAGAGTCC (SEQ ID NO: 68) KIAA0196x17RGTAAGGTCTTGTTCGCGATAGGTT (SEQ ID NO: 69) KIAA0196x18FAGAACGAATAGTTGACAATCTACTC (SEQ ID NO: 70) KIAA0196x18RTGAGGTTTGGGATGTGTACTCTAA (SEQ ID NO: 71) KIAA0196x19FAATTATATGGAAAAGGGATAACTAGGT (SEQ ID NO: 72) KIAA0196x19RTAAAGGGTCAGAATATGAGTTGACAAG (SEQ ID NO: 73) KIAA0196x20FTTGGTGCCGCATGTCCTGTTGAGTC (SEQ ID NO: 74) KIAA0196x20RAAGTCTTATCTTCCCAAGTTGAAAC (SEQ ID NO: 75) KIAA0196x21CCCAGCCTCTGTTCTGCATAGCAT (SEQ ID NO: 76) and 22F KIAA0196x21AAGAACAGATCCAGAAACGGAGAT (SEQ ID NO: 77) and 22R KIAA0196x23FAAGGCCCAGTGAAGAATTGTCATC (SEQ ID NO: 78) KIAA0196x23RCTGAAGAAACTGGGGTGCGTAGAT (SEQ ID NO: 79) KIAA0196x24FCTGAGGCTTGAAAAGATTACATCAC (SEQ ID NO: 80) KIAA0196x24RCTTCCCCTTTGTCATGAGCTTTCAC (SEQ ID NO: 81) KIAA0196x25FTCCCACACTCCCCCTATATTCACCTC (SEQ ID NO: 82) KIAA0196x25RAGAAAAGATCTCATATCCGACATAGG (SEQ ID NO: 83) KIAA0196x26FGACCCCTGGAATGCCCTACCAATC (SEQ ID NO: 84) KIAA0196x26RCTGGCAGGGTGACTAAGGATGGAC (SEQ ID NO: 85) KIAA0196x27FGATAGATAGCAGGGATCGTGTTGT (SEQ ID NO: 86) KIAA0196x27RAGGCATCTACTGTGAACGACCTAT (SEQ ID NO: 87) KIAA0196x28FAAAGGGGCTGTTTCAAGGAGTCG (SEQ ID NO: 88) KIAA0196x28RAGTTTTTGAATCATAAGCGAGACG (SEQ ID NO: 89)

PCR was performed using 50 ng DNA, 20 pmol of each primer, 10× buffer,0.25 nM dNTPs and 0.15 ul of Taq (Qiagen). Initial denaturation for 5minutes at 94° C. was followed by 30 cycles of 30 seconds denaturationat 94° C., 30 seconds annealing at 55° C. (for all exons except forexons 15 and 26), and 45 seconds elongation at 72° C. A final extensionat 72° C. was performed for 7 minutes. For exon 15, a 50° C. annealingtemperature was used, and for exon 26, 10 cycles of a touchdown reactionwere performed from 68° C.-63° C., followed by 25 cycles at 63° C.

Variants were first tested in 12 controls by sequencing, followed byallele-specific oligomerization (ASO).^(19,20) Briefly, 4 μl of PCRproduct was hybridized onto Hybond-N+™ Nylon membranes (AmershamBiosciences) using a dot blot apparatus. P-32-labelled probes specificto the mutation or normal sequence were hybridized then visualized onautoradiographic film after overnight exposure. ASO primers for exon 11are 5′-ACTAGAAAACCTTCAAGCT-3′ (SEQ ID NO: 90) (normal) and5′-ACTAGAAGACCTTCAAGCT-3′ (SEQ ID NO: 91) (mutated). For exon 14, ASOprimers of 5′-GGAGAGTTGGTATC-3′ (SEQ ID NO: 92) (normal) and5′-GGAGAGTTCGTATC-3′ (SEQ ID NO: 93) (mutated) were used. Exon 15 ASOprimers were 5′-CACTGAAGGTTTTG-3′ (SEQ ID NO: 94) (normal) and5′-CACTGAAGTTTTTG-3′ (SEQ ID NO: 95) (mutated).

Protein Sequence Alignment

Cluster analysis was performed using the Probcons™ v. 1.09 program.Proteins from aligned species included Homo sapiens (Q12768) (SEQ ID NO:26), Canis familiaris (XP_(—)532327) (SEQ ID NO: 30), Pan troglodytes(XP_(—)519952) (SEQ ID NO: 27), Drosophila melanogastar (CG12272) (SEQID NO: 33), Caenorhabditis elegans (CE13235) (SEQ ID NO: 35), Xenopustropicalis (MGC89323) (SEQ ID NO: 32), Rattus norvegicus (XP_(—)343250)(SEQ ID NO: 29); Danio rerio (BC045490) (SEQ ID NO: 34), Gallus gallus(XP_(—)418-441) (SEQ ID NO: 31), Dictyostelium discoideum (EAL63144)(SEQ ID NO: 36), and Mus musculus (NP_(—)705776.2) (SEQ ID NO: 28) (SeeFIG. 16 for alignment).

Homology Modeling

The size of the strumpellin protein (1159aa) made it prohibitive toobtain a template for the entire protein. Instead, 200 amino acidsaround the two mutations were selected (aa 501-725) and inputted in thePhyre™ program version 2.0³⁹. The template with the highest score wasselected, namely 1dn1b from the Neuronal-Sec1 Syntaxin 1a complex. TheSwissProt™ database viewer version 3.721 was used to visualize the modelconcentrating on the alpha helix in which the two mutations lie and on asecond alpha helix nearby in 3D space (See FIG. 5). Peptidesincorporating one or the other identified point mutation were visualizedin the same manner.

Expression Studies

Northern Blot and RT-PCR Analysis

The KIAA0196 cDNA pBluescript™ clone was kindly provided by the KazusaDNA Research Institute. A 1 kb probe specific to the c-terminal regionof strumpellin was generated by digesting the KIAA0196-pBluescript™vector with XhoI and NotI. 30 ug of total RNA per sample was loaded. RNAwas extracted from various regions of the brain of a control individual.A reverse-transcriptase reaction was performed using MMLV-RT(Invitrogen). Primers in exons 10 (Forward) and 15 (Reverse) of KIAA0196were used as described in Table 1 above. GAPD cDNA was amplified as acontrol.

Constructs

Each mutation was introduced into the KIAA0196 pBluescript™ clone bysite-directed mutagenesis using the primers5′-CTGGAGAGTTCGTATCCTATGTG-3′ (SEQ ID NO: 96) for the exon 14 variantand 5′-CCTATGTGAGAAAATTTTTGCAGATC-3′ (SEQ ID NO: 97) for the exon 15variant, along with primers of their complementary sequence. Wildtypeand mutant KIAA0196 cDNAs were cloned, upstream of a Myc and His tags,into a pCS2 vector and transcribed in vitro using the SP6 mMESSAGEmMachine™ kit (Ambion) for zebrafish studies. The protein expressionfrom each these constructs was validated following their transientexpression in cell culture (HeLa) and subsequent western blot analysiswith an anti-Myc antibody. A band at the appropriate height (˜134 kDafor KIAA0196) was observed (See FIG. 7).

Zebrafish Knockdown Studies

Morpholino Injections

Wildtype zebrafish were raised and mated as previously described.²²

Antisense morpholinos (AMO) were designed and purchased from GenetoolsLLC (Philomath, Oreg.). The morpholino sequences were designed againstthe zebrafish strumpellin ortholog, BC045490. The oligonucleotide,CTCTGCCAGAAAATCAC[CAT]GATG (SEQ ID NO: 98) (KIAA MO) binds to the ATG ofthe KIAA0196 gene preventing its translation andCTCTcCCAcAAAATgAg[CAT]cATG (SEQ ID NO: 99) (CTL MO) is a five base pairmismatch control. AMO injections were performed as previously describedat a concentration of 0.8 mM.23 The rescue injections were performed asmentioned above with a morpholino and mRNA concentration of 0.8 mM and50 ng/ul respectively.

Immunohistochemistry

Standard protocols were used for immunohistochemistry.²² Briefly, threeday old embryos were fixed in 4% paraformaldehyde, washed, and blockedat room temperature. Primary antibodies [anti acetylated tubulin, 1:50(Sigma)] were added overnight. After extensive washing, the embryos wereincubated with the fluorescently labelled secondary antibody Alexa 568(Molecular Probes). Imaging was performed on an UltraView™ LCI confocalmicroscope (Perkin Elmer) using Methamorph™ Imaging software (UniversalImaging Corporation). The statistical significance between the differentconditions was calculated using a chi square test.

Clinical Information and Family Details

The SPG8 family FSP24 is from the province of British Columbia, Canada.It is composed of 13 members affected with a spastic gait and lower limbstiffness, 10 of which have been collected (See FIG. 1 a). Symptoms werefirst observed in individuals between the ages of 35 and 53.Intrafamilial phenotypic heterogeneity exists as noted by the symptomspresented and the range in disease severity in patients. Deep tendonreflexes were brisk or increased, and decreased vibration sensation wasalso noted in three patients. Occasional bladder control problems werealso observed. Walking aids were required for some individuals while oneis confined to a wheelchair. Together, these features are consistentwith a pure, uncomplicated HSP similar to that described for otherfamilies linked to the SPG8 locus. Family FSP29 is of European descentresiding in the United States. There are 31 affected individuals in thefamily, and 10 have been collected (See FIG. 1 b). Age of onset wasquite variable with symptom onset ranging in patients from theirtwenties to their sixties. The family was negative for mutations in thespastin gene.

Example 2 Linkage Analysis

In FSP24, seven markers spanning the candidate region from markersD8S586 to D8S1128 were genotyped in the 10 affected individualscollected (FIG. 1 a). The genotyping and locus exclusion were performedas described in Example 1. A disease haplotype segregated with thedisease in all 10 affected individuals (See Table 2 below). Arecombination event occurred in one individual (FIG. 1 a) betweenmarkers D8S586 and D8S1804 defining the proximal border of the locus inthis family. No lower recombinant was identified nor searched for sincethe haplotype extended beyond the limits of the SPG8 locus. The maximumLOD score for this family was 3.43 at θ=0 using CEPH allele frequenciesfor the marker D8S1804, along with a maximum multipoint of 4.20 atmarker D8S1799.

TABLE 2 Haplotype comparison between SPG8 linked families MarkerPosition (Mb) FSP24 FSP29 FSP34 D8S586 121.2 1 11  11  D8S1804 124.8 5 33 D8S1832 125.4 2 2 NT D8S1179 125.9 3 9 9 KIAA0196 126.1 L619F L619FL619F rs2293890 126.4 G C C D8S1774 127.5 3 5 4 D8S1128 128.5 7 5 1Flanking markers in this candidate region are D8S1832 and D8S1774 forfamily FSP29. NT = not typed

The same seven markers tested in FSP24 were genotyped for FSP29. Adisease haplotype was established for all 10 collected affectedindividuals that included many informative recombination events. Theproximal recombinant occurred between markers D8S1799 and D8S1832 inthree affected individuals (FIG. 1 b), and the distal recombinant wasbetween markers D8S1774 and D8S1128 for another affected individual(FIG. 1 b). This yielded a candidate interval of 3.15 Mb. The maximumLOD score for this family was 5.62 (θ=0) for the marker D8S1179 whenusing CEPH allele frequencies. Multipoint analysis was also conductedfor this family in this region yielding a maximum LOD score of 6.73, 0.5cM centromeric to the D8S1128 marker.

Example 3 Gene Screening and Mutation Analysis

The previously published SPG8 locus spanned 3.4 cM (1.04 Mb) betweenmarkers D8S1804 and D8S1179 on chromosome 8q23-8q24. Nine known geneswere screened surrounding this candidate region as annotated in the UCSChuman genome browser (UCSC golden path, http://www.genome.ucsc.edu/⁴⁰)May 2004 update along with many clustered ESTs and mRNAs that aligned tothe locus without detecting a mutation. It was Therefore opted toredefine the candidate region based on the critical interval determinedby an upper recombinant in the FSP29 family at the marker D8S1832 and alower recombinant at D8S1774 was based on published data (FIG. 2 a).¹⁴This increased the size of the region to 5.43 cM (3.15 Mb), whichcontains 3 additional known genes. In total, 12 genes were sequencedbetween markers D8S1804 and D8S1174 (FIG. 2 b). These additional geneswere screened and three mutations were identified in the KIAA0196 geneusing the mutation screening method described in Example 1 above (FIG.2C).

A valine-to-phenylalanine mutation was identified in amino acid 626 forfamilies FSP24 and FSP29 (p.V626F) (FIG. 3 a). All affected individualscollected from each family were screened and were positive for thismutation. This G to T nucleotide change is at position 2205 of the mRNA(see FIG. 14). A total of 500 ethnically matched control individuals(400 from North America, and 100 from CEPH) were negative for thismutation after screening by a combination of allele-specificoligomerization (ASO) and sequencing. No unaffected members and spousecontrols in any family were positive for the mutation in exon 15. Thefamily previously described by Reid et al.¹⁵ was also screened forKIAA0196 and the V626F mutation was identified.

A second mutation was identified in the Brazilian family¹⁶ in exon 14, aG to C transition at position 2186 of the mRNA (FIG. 3 b). Thisleucine-to-phenylalanine change (p.L619F) is only 7 amino acids awayfrom the V626F mutation. It was also not found in 500 controls usingASO.

The KIAA0196 gene was screened in probands from 24 additional dominantHSP families that are negative for mutations in both spastin andatlastin, resulting in the identification of two more families withmissense mutations in the KIAA0196 gene. Thus, the frequency ofmutations in SPG3A and SPG4-negative autosomal dominant cohort is ˜8% (2in 24). FSP34 has the same p.V626F change in its 3 affected collectedmembers. This family is originally from Great Britain, residing inCanada (FIG. 1 c). Haplotype analysis of this family with markersD8S1804, D8S1179, D8S1774 and D8S1128 indicated that there is allelesharing between this family and family FSP29 suggesting an ancestralhaplotype (Table 2 above). An additional mutation was found in exon 11in three affected siblings of another North American family of Europeanorigin, FSP91 (FIG. 1 d). This c.A1740G transition results in anasparagine to aspartate amino acid change (p.N471D), and is not presentin the 500 controls tested (FIG. 3 c). The Hedera et al. family¹⁴ wasnot screened but it is expected that affected members possess a mutationin the KIAA0196 genes.

Protein sequence alignment was performed as described in Example 1above. Mutated amino acids at positions 619 and 626 are strictlyconserved across all eleven species examined all the way to the socialamoeba, Dictyostelium discoideum (FIG. 3D). Indeed, the entire regionsurrounding these two mutations appears to be functionally relevant forthe protein as 73 consecutive amino acids (aa 576-649) are 100%identical between the human, dog, chicken, mouse, rat and orangutan.Despite this high level of conservation, this region is not a knowndomain, based on NCBI's conserved domain database search, NCBI's BLAST™search, and the Sanger Institute's Pfam database.⁴¹ Position 471 isconserved across all species save for Drosophila melanogastar with aglutamine residue and Xenopus tropicalis with a histidine.

The exon 15 mutation is in the very first nucleotide of the exon, whichleads to the speculation that the splicing of this exon might becompromised in these families. Splice site prediction programs includingNetGene2™ suggested that the strength of the splice site acceptor may bereduced by 33% in the mutant form.⁴² However, both normal and mutantalleles were observed in cDNA analysis of patient lymphoblasts usingseveral pairs of primers. The KIAA0196 gene was expressed ubiquitously,including all regions of the brain which were examined by RT-PCR (FIG. 3e). There were no alternative splice isoforms detected in control brainsamples and the patient whole blood samples by RT-PCR and northernanalysis (FIGS. 3 e and 3 f). For the full KIAA0196 gene, all splicedESTs and mRNAs from the UCSC browser, May 2004 draft, were analyzed forpotential alternative splice products. One alternative first exon oftenappears; however, out of the 356 entries, only two (AK223628 andDA202680) contain exons which are skipped. Thus overall, the gene is notfrequently spliced, and the two spliced entries may represent spurioustranscripts.

Example 4 KIAA0196 Profile

The KIAA0196 gene spans 59.7 kilobase pairs of genomic DNA, is 28 exonslong and codes for a protein of 1159 amino acids that is namedstrumpellin herein. The EBI institute's InterPROScan™ program⁴³predicted a spectrin-repeat containing domain from amino acids 434 to518. Thus, the mutation at position 471 may abrogate the binding of thespectrin domain with other spectrin-repeat containing proteins. Inexamining the secondary structure using PSIPRED²⁴, 74% of the protein isconsidered to be alpha-helical. The program further predicted an α-helixin the protein from amino acids 606 to 644, encompassing the two othermutations which have been identified. The helix consists of a heptamericrepeat with hydrophobic residues aligning in inaccessible regions at thecenter of the helix. The hydrophobic lysine and valine amino acids areseven amino acids apart in the protein sequence; thus it is expectedthey would be buried in the helix, close in 3D space (FIG. 5 a).

Human KIAA0196 gene is known to have previously been implicated inprostate cancer.³² An increase in gene copy number was assayed byreal-time quantitative PCR and fluorescence in-situ hybridization,determining over ten-fold overexpression of the gene in PC-3 prostatecancer lines, and in ˜⅓ of advanced prostate cancers examined.³²

Analysis of other species has provided some insight into a potentialfunction for KIAA0196. A 118 kDa homologue of the strumpellin proteinwas identified as part of a TATA-binding protein-related factor 2 (TRF2)complex in a Drosophila nuclear extract.³³ Eighteen proteins were pulleddown along with TRF2 in this complex including NURF and SWI, withfunctions for chromatin remodeling and transcription activation. TRF2 isselective for promoters lacking TATA or CAAT boxes. One protein of thecomplex is DREF which binds to DRE elements common in controlling genesinvolved in cell cycle regulation and cell proliferation.^(34,35)

Example 5 Homology Modeling

Homology modeling was performed as described in Example 1 above. Giventhe high proportion of KIAA0196 considered to be alpha-helical, it isnot surprising that the optimal homology modeling candidates are similarin secondary structure composition.

This is true for 1dn1b, a stat-like t-SNARE protein neuronal-Sec1Syntaxin 1a complex. This is the most appropriate model for strumpellinaccording to the Phyre™ program. The two mutated residues lie within analpha-helix from amino acids 619-628 which is in close 3D proximity toanother alpha-helix from residues 665-670 (FIG. 4 a). A mutation ineither Val-626 or Leu-619 to a phenylalanine residue would appear tohave significant structural implications given the change in bulkinessbetween each of the residues. In addition, Tyr-622 points in the samedirection from the alpha-helix residue. To have two amino acids witharomatic rings in such a physical proximity could force apart thealignment of the two alpha-helices or induce alterations in thealpha-helix backbone

The one known domain in strumpellin is a spectrin repeat which consistsof three α-helices of a characteristic length wrapped in a left-handedcoiled coil.²⁶ These spectrin repeats appear in thespectrin/dystrophin/α-actinin family. The spectrin proteins havemultiple copies (15-20) of this repeat which can then form multimers inthe cell. Spectrin also associates with the cell membrane via spectrinrepeats in the ankyrin protein. Likewise, four spectrin repeats arefound in α-actinin beside two N-terminal calponin homology domains whichanchor the complex to actin.²⁷ This effectively connects the cellmembrane with the actin cytoskeletal network. The stability andstructure of this network also provides appropriate routes forintracellular vesicular transport, a mechanism already linked to othermutated HSP genes. Proteins with three spectrin repeats or fewer can beconsidered to have transient association with the spectrin network. Thesingle repeat in strumpellin is more likely to be involved in dockingwith one of the cytoskeletal spectrin repeats, which could help inprotein localization or signal transduction.

Proteins with a spectrin repeat have been identified in otherneurological disorders, most notably dystrophin, mutated in myotonicdystrophy (MIM300377).28 The repeat also has been found in a form ofcerebellar ataxia (MIMI17210).²⁹ β-III spectrin itself is found to bemutated in SCA5.³⁰ While none of the genes mutated in HSP have aspectrin domain, L1CAM (SPG1) has an indirect association.^(9,31) L1CAMis a single-pass transmembrane protein with a glycosylated extracellularcomponent which facilitates the outgrowth and migration of neurons inthe corticospinal tract. The intracellular c-terminus however binds tothe spectrin-repeat containing protein, ankyrin, linking the cellmembrane to intracellular spectrin. Thus, strumpellin with its spectrindomain may also be involved in this process.

Example 6 Zebrafish Rescue Experiments

In order to validate the functional phenotype of SPG8 mutations in vivo,a zebrafish model was developed. Morpholino oligonucleotide knockdown ofthe KIAA0196 protein ortholog in zebrafish (KIAAMO) was performed asdescribed in Example 1 above. It resulted in an enlarged heart cavityalong with a curly tail phenotype which severely impaired the ability ofthe fish to swim properly. The overall phenotype ranged in severity andwas classified in 3 major groups: normal, slightly curly, and severelycurly. This phenotype was clearly visible after dechorionating by 1 daypost fertilization (dpf). At 3 dpf, wildtype zebrafish are ˜5 mm longwith a straight tail (FIG. 5A). Fish injected with a mismatch-controlmorpholino (CTLMO) were initially used to titer a KIAAMO specificnon-toxic injection dose (FIG. 5 b). Injection of the KIAAMO resulted in66 of 178 (37%) severely curly fish and 50 of 178 (28%) slightly curlyfish (See Table 3 below and FIGS. 5 c and d).

TABLE 3 Phenotype profile from zebrafish morpholino oligonucleotideknockdown expressed in percent (total number) Condition Normal Slightcurve Severe curve Dead Total KIAA0196 morpholino 19.1 (34) 28.1 (50)37.1 (66) 15.7 (28) 178 Control morpholino 56.1 (83) 24.3 (36)  7.4 (11)12.2 (18) 148 Wildtype rescue  63.2 (127) 19.4 (39)  8.0 (16) 9.5 (9)201 Mutant x14 rescue 16.0 (32) 37.0 (74) 36.0 (72) 11.0 (22) 200 Mutantx15 rescue 13.2 (29) 37.4 (82) 30.1 (66) 19.2 (42) 219

The KIAAMO fish had a significantly different distribution of phenotypicgroups compared to CTLMO injections (p<0.001). When wildtype humanKIAA0196 mRNA was co-injected with KIAAMO, the curly tail phenotype wasrescued to levels comparable to CTLMO injections (p=0.51) (FIGS. 5 e andf). This suggests that in zebrafish, human KIAA0196 mRNA can compensatefor the loss of endogenous zebrafish mRNA. Conversely, co-injection ofhuman KIAA0196 mRNA incorporating either the exon 14 or exon 15 mutationfailed to significantly rescue the phenotype (FIG. 5G, H). Injection ofmutant exon 14 or exon 15 mRNA alone (without morpholinos) did not leadto curly tail phenotype or influence lethality in zebrafish, suggestingthat the two mutations do not exert a dominant negative effect.Approximately 200 embryos were injected per experimental condition(Table 3 above). The difference in distribution between KIAAMO injectionalone and KIAAMO co-injection with wildtype mRNA was significant(p<0.001). Similarly, co-injection of wildtype mRNA versus either exon14 or exon 15 mutant mRNA was significantly different with a p-value<0.001. There was no statistical difference between the co-injection ofthe exon 14 mutant and the exon 15 mutant (p=0.10).

Upon histochemical analysis of the embryos using an anti-acetylatedtubulin stain for growing axons using the method described in Example 1above, it was found that the motor neurons in the spinal cord did notdevelop normally (FIG. 6). Motor neuron axons in fish injected withKIAAMO alone or with the mutant mRNAs were shorter and showed abnormalbranching. The structure of interneurons in the spinal cord was alsodifferent. The absence of the KIAA0196 gene or mutations in this geneduring early development thus seemed to hamper axonal outgrowth.

Example 7 Production of Antibodies

Three different peptides corresponding to amino acids 62-76(KGPELWESKLDAKPE (SEQ ID NO: 100), 652-669 (VPTRLDKDKLRDYAQLGP (SEQ IDNO: 37)) and 1132-1147 (YVRYTKLPRRVAEAHV (SEQ ID NO: 101)) weresynthesized. The sequence of the first peptide (62-76) corresponds toprotein residues present near the amino terminal portion of strumpellin.The sequence of the second peptide (652-669) corresponds to residuesfound in the middle of the protein. Finally, the sequence of the thirdpeptide (1132-1147) corresponds to residues near the carboxy terminalportion of the protein.

Every month a dose of each peptide was injected to two separate rabbits.These intraperitoneal injections were carried over for a period of fivemonths. A month following each injections, blood samples were collectedfrom each animals and the cellular fraction removed the sera were storedat −80° C. Following the recovery of the last samples where a completefinal bleed was achieved, all the animals were euthanized. The sera fromthe second peptide (652-669) were observed to be the most specific forstrumpellin (FIG. 7 b).

Example 8 Detecting KIAA0196 Mutations in a Subject Sample

The method for detecting a mutation in the KIAA0196 gene involves theamplification of a patient's DNA by the Polymerase Chain Reaction (PCR)using primers designed to specifically recognize flanking genomicsequences of the KIAA0196 gene, such as those listed in Table 1 above. Aseries of PCR amplifications are necessary to cover the entire codingregions of KIAA0196 and its flanking splice site regions. The product ofthese amplifications are subsequently sequenced and examined for thepresence of mutations using appropriate software (e.g. the SeqMan™program from the DNASTAR™ sequence analysis package). Sequences of thepatient's DNA amplifications are compared to a reference sequence whereno mutation can be found and or to the reference sequences fromdatabases like UCSC.

The optimal PCR reactions for the amplification of KIAA0196 are thefollowing: 50 ng DNA, 20 pmol of each oligonucleotide primer, 10×buffer, 0.25 nM dNTPs and 0.15 ul of Taq (Qiagen). An initialdenaturation step of 5 minutes at 94° C. is done and it is followed by30 cycles of 30 seconds denaturation at 94° C., 30 seconds annealing at55° C. (for all exons except for exons 15 and 26), and 45 secondselongation at 72° C. A final extension at 72° C. of 7 minutes is finallyperformed. In the case of exon 15, a 50° C. annealing temperature wasused, and in the case of exon 26, 10 cycles of a touchdown reaction wereperformed from 68° C.-63° C., followed by 25 cycles at 63° C.

To identify the three specific mutations of the present invention, threeseparate PCRs were performed using oligonucleotide primers thatcorresponded to the sequences surrounding exons 11, 14, and 15 with thepatients DNA. Following the sequencing of these products, the sequencetraces generated by the software were analyzed visually.

Example 9 Detecting Mutant KIAA0196 RNA in a Subject Sample

To detect the presence of mutant RNA, PCR reactions may be performedusing oligonucleotide primers specific to the cDNA sequence (codingsequences of DNA exclusively, not sequences flanking the differentcoding regions) of KIAA0196, such as those in Table 4 below. RNA needsto be extracted from patients using standard methods, and areverse-transcription PCR (RT-PCR) is performed. The cDNA productsgenerated by this reaction are then used as a template for the PCRamplifications. The sequence trace results are then analyzed usingappropriate software for the detection of mutations. The type of proteinmodifications the occurrence of any mutation within the RNA (hererepresented by the cDNA) can be predicted using a table listing theamino acids produced by the different codons possible.

TABLE 4 Primers for cDNA analysis of the KIAA0196 gene KIAA0196rna1FCCGGGACTGCGGATAGAAGA (SEQ ID NO: 102) KIAA0196rna1RAATCCTGTAGCTCTGGCTTAGCATC (SEQ ID NO: 103) KIAA0196rna2FTCTGAGTTTATTCCTGCTGTGTTCA (SEQ ID NO: 104) KIAA0196rna2RCTCTCGGGATAGTTGGATGGTCTTT (SEQ ID NO: 105) KIAA0196rna3FGCTGCTCGATCTTCTGCTGATTCAA (SEQ ID NO: 106) KIAA0196rna3RATCGGATGGCAACATTGCAGTCTCT (SEQ ID NO: 107) KIAA0196rna4FTAAGGGAGGAGATGGTTCTGGACA (SEQ ID NO: 108) KIAA0196rna4RTCGAGTATCGGCAAGAAACTGACA (SEQ ID NO: 109) KIAA0196rna5bFTGAAACCCCTAACCAGAGTGGAGAAA (SEQ ID NO: 110) KIAA0196rna5RATCGTGGGCCTAGCTGAGCATAGT (SEQ ID NO: 111) KIAA0196rna6FAGCTTCAGACCCACGACATTATTG (SEQ ID NO: 112) KIAA0196rna6RCCACAGGGGTAAACTTGGGTATTG (SEQ ID NO: 113) KIAA0196rna7FTTGGCAAAGCATGTACCAGTCCAC (SEQ ID NO: 114) KIAA0196rna7RTCTGCATCTGCCCAACCTTCATTA (SEQ ID NO: 115) KIAA0196rna8FTCCGCCATTGCCAAAACACAGA (SEQ ID NO: 116) KIAA0196rna8RGGCCAATCAGCGCCAGGAACT (SEQ ID NO: 117) KIAA0196rna9FGCTTGTCCTGGGACTGCTCACTC (SEQ ID NO: 118) KIAA0196rna9RAGAGGCAGTACAAAAATGTGTTCT (SEQ ID NO: 119)

Example 10 Assay to Detect Mutation in Subject Sample

A Western blot is performed using an antibody specific to the KIAA0196product and protein lysates prepared from patient tissue orlymphoblastoid cell lines to detect mutant protein in a patient'sbiological sample.

Example 11 Identifying Strumpellin-Interacting Proteins

Protein-protein protocols such as the yeast two hybrid (Y2H), GSTpulldown and co-immunoprecipitation approaches are used to identifystrumpellin-interacting proteins. These three approaches are performedusing both normal and mutant strumpellin to establish which interactionsmay be specific to the mutated forms of the protein, but also to see insome interactions with the normal form of the protein decrease orincrease with the mutated proteins. Such interactions with the mutatedproteins are to be investigated only in the event that these are stablyexpressed. Interactions occurring through the spectrin domain ofstrumpellin in particular is examined.

Although the present invention has been described hereinabove by way ofspecific embodiments thereof, it can be modified, without departing fromthe spirit and nature of the subject invention as defined in theappended claims.

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1. A method for diagnosing the presence of SPG8-associated hereditaryspastic paraplegia (HSP) or predicting the risk of developingSPG8-associated HSP in a human subject, comprising, a) obtaining anucleic acid sample from a subject suspected of having HSP or being atrisk of developing HSP; and b) determining the presence or absence of anucleotide sequence defect in all or a portion of, a nucleic acidsequence encoding a polypeptide consisting of the sequence of SEQ ID NO:19 in the nucleic acid sample obtained from the subject; wherein thedefect is a missense mutation, a splice site mutation, a nonsensemutation, an insertion, a deletion or a frameshift mutation; wherein themutation causes a disruption of the function or conformation of theencoded protein, or decreased expression or absence of the encodedprotein; and wherein detection of a sequence defect is indicative thatthe subject has or is at risk of developing SPG8-associated HSP.
 2. Themethod of claim 1, wherein said sample comprises DNA.
 3. The method ofclaim 1, wherein said sample comprises RNA.
 4. The method of claim 1,wherein the sequence comprises a mutation in the nucleic acid sequenceresulting in a mutant polypeptide in which at least one amino acidresidue of SEQ ID NO: 19 is substituted with another amino acid residue,and wherein the at least one amino acid residue is selected from thegroup consisting of an asparagine residue at position 471, a leucineresidue at position 619 and a valine residue at position
 626. 5. Themethod of claim 1, wherein the sequence comprises a mutation in the generesulting in a mutant polypeptide in which amino acid residue 471 of SEQID NO: 19 is substituted with an aspartate residue, or in which aminoacid residue 619 of SEQ ID NO: 19 is substituted with a phenylalanineresidue or in which amino acid residue 626 of SEQ ID NO: 19 issubstituted with a phenylalanine residue.
 6. The method of claim 1,wherein the sequence comprises a mutation in the gene, wherein the geneis as set forth in SEQ ID NO: 18 the mutation being selected from thegroup consisting of a substitution of a guanine at position 2205 withanother nucleotide, a substitution of a guanine at position 2186 withanother nucleotide, and a substitution of an adenine at position 1740with another nucleotide.
 7. The method of claim 1, wherein the sequencecomprises a mutation in the gene, wherein the gene is as set forth inSEQ ID NO: 18 the mutation being selected from the group consisting of asubstitution of a guanine at position 2205 with a thymine, asubstitution of a guanine at position 2186 with a cytosine, and asubstitution of an adenine at position 1740 with a guanine.
 8. A methodof detecting the presence or absence of a nucleotide sequence mutationin the coding sequence of a strumpellin gene, said method comprising thesteps of: a) obtaining and analyzing a nucleic acid test samplecontaining all, or a portion of, the strumpellin gene coding sequence;b) comparing the results of said analysis of said sample of step a) withthe results of an analysis of a control nucleic acid sample containing awildtype strumpellin gene coding sequence, wherein the wildtypestrumpellin gene coding sequence encodes a polypeptide comprising thesequence of SEQ ID NO: 19; and c) determining the presence or absence ofat least one nucleotide sequence difference in the coding sequence ofthe strumpellin gene of the test sample as compared to the wildtypecoding sequence of the strumpellin gene; wherein detection of a sequencedifference is indicative of a mutation in the coding sequence of thestrumpellin gene of the test sample.
 9. The method of claim 8 whereinthe nucleic acid sample is amplified prior to analysis.
 10. The methodof claim 8 wherein the mutation is a missense mutation, a splice sitemutation, a nonsense mutation, an insertion, a deletion or a frameshiftmutation, wherein the mutation causes a disruption of the function, aconformation of the encoded protein, or decreased expression or absenceof expression of the encoded protein.
 11. The method of claim 8, whereinthe mutation results in a mutant polypeptide in which at least one aminoacid residue of SEQ ID NO: 19 is substituted with another amino acidresidue, and wherein the at least one amino acid residue is selectedfrom the group consisting of an asparagine residue at position 471, aleucine residue at position 619 and a valine residue at position 626.12. The method of claim 8, wherein the mutation results in a mutantpolypeptide in which amino acid residue 471 of SEQ ID NO: 19 issubstituted with an aspartate residue, or in which amino acid residue619 of SEQ ID NO: 19 is substituted with a phenylalanine residue or inwhich amino acid residue 626 of SEQ ID NO: 19 is substituted with aphenylalanine residue.
 13. The method of claim 8, wherein the genecoding sequence comprises SEQ ID NO: 18 and, the mutation is selectedfrom the group consisting of a substitution of a guanine at position2205 with another nucleotide, a substitution of a guanine at position2186 with another nucleotide, and a substitution of an adenine atposition 1740 with another nucleotide.
 14. The method of claim 8,wherein the coding sequence comprises SEQ ID NO: 18 and the mutation isselected from the group consisting of a substitution of a guanine atposition 2205 with a thymine, a substitution of a guanine at position2186 with a cytosine, and a substitution of an adenine at position 1740with a guanine.
 15. The method of claim 8, wherein the analysiscomprises one or more methods selected from the group consisting of:sequence analysis; a fragment polymorphism assays; a hybridizationassays and computer based data analysis.
 16. A method of detecting thepresence or absence of a nucleotide sequence mutation in a strumpellingene coding sequence, said method comprising the steps of: a) obtainingand analyzing a nucleic acid test sample containing all, or a portionof, the coding sequence; b) comparing the results of said analysis ofsaid sample of step a) with the results of an analysis of a controlnucleic acid sample containing a wildtype strumpellin gene codingsequence, wherein the wildtype strumpellin gene coding sequencecomprises the SEQ ID NO: 18; and c) determining the presence or absenceof at least one nucleotide sequence difference in the strumpellin genecoding sequence of the test sample as compared to the wildtypestrumpellin gene coding sequence, wherein the detection of a sequencedifference is indicative of a mutation in the strumpellin gene codingsequence of the test sample.
 17. The method of claim 16 wherein thenucleic acid sample is amplified prior to analysis.
 18. The method ofclaim 16, wherein the mutation is selected from the group consisting ofa substitution of a guanine at position 2205 with another nucleotide, asubstitution of a guanine at position 2186 with another nucleotide, anda substitution of an adenine at position 1740 with another nucleotide.19. The method of claim 16, wherein the mutation is selected from thegroup consisting of a substitution of a guanine at position 2205 with athymine, a substitution of a guanine at position 2186 with a cytosine,and a substitution of an adenine at position 1740 with a guanine.
 20. Amethod for diagnosing the presence of SPG8-associated hereditary spasticparaplegia (HSP) or predicting the risk of developing SPG8-associatedHSP in a human subject, comprising, a) obtaining a nucleic acid samplefrom the subject suspected of having HSP or being at risk of developingHSP; and b) determining the presence or absence of a nucleotide sequencedefect in all, or a part of, a sequence encoding a polypeptidecomprising the sequence of SEQ ID NO: 19 in the nucleic acid sampleobtained from the subject, wherein the defect comprises a mutationresulting in a mutant polypeptide in which at least one amino acidresidue of SEQ ID NO: 19 is substituted with another amino acid residue,and wherein the at least one amino acid residue is selected from thegroup consisting of an asparagine residue at position 471, a leucineresidue at position 619 and a valine residue at position 626, wherebythe detection of the defect is indicative that the subject has or is atrisk of developing SPG8-associated HSP.
 21. The method of claim 20wherein determining the presence or absence of a nucleotide sequencedefect in of the nucleic acid sample includes an amplification step. 22.A method for diagnosing the presence of SPG8-associated hereditaryspastic paraplegia (HSP) or predicting the risk of developingSPG8-associated HSP in a human subject, comprising, a) obtaining anucleic acid sample from the subject suspected of having HSP or being atrisk of developing HSP; and b) determining the presence or absence of anucleotide sequence defect in all, or a part of, a sequence encoding apolypeptide comprising the sequence of SEQ ID NO: 19 in the nucleic acidsample obtained from the subject, wherein the defect comprises amutation in the gene coding sequence resulting in a mutant polypeptidein which amino acid residue 471 of SEQ ID NO: 19 is substituted with anaspartate residue, or in which amino acid residue 619 of SEQ ID NO: 19is substituted with a phenylalanine residue or in which amino acidresidue 626 of SEQ ID NO: 19 is substituted with a phenylalanineresidue, detection of the defect is indicative that the subject has oris at risk of developing SPG8-associated HSP.
 23. The method of claim 22wherein the determining the presence or absence of a nucleotide sequencedefect in the nucleic acid sample indicates an amplification step.
 24. Amethod for diagnosing the presence of SPG8-associated hereditary spasticparaplegia (HSP) or predicting the risk of developing SPG8-associatedHSP in a human subject, comprising, a) obtaining a nucleic acid samplefrom the subject suspected of having HSP or being at risk of developingHSP; and b) determining the presence or absence of a sequence defect inall, or a part of, a coding sequence encoding a polypeptide comprisingthe sequence of SEQ ID NO: 19, in the nucleic acid sample obtained fromthe subject, wherein the defect comprises a mutation in the codingsequence, wherein the coding sequence is as set forth in SEQ ID NO: 18the mutation being selected from the group consisting of a substitutionof a guanine at position 2205 with another nucleotide, a substitution ofa guanine at position 2186 with another nucleotide, and a substitutionof an adenine at position 1740 with another nucleotide, whereby thedetection of the defect is indicative that the subject has or is at riskof developing SPG8-associated HSP.
 25. The method of claim 24 whereinthe determining the presence or absence of a defect in the nucleic acidsample includes an amplification step.
 26. A method for diagnosing thepresence of SPG8-associated hereditary spastic paraplegia (HSP) orpredicting the risk of developing SPG8-associated HSP in a humansubject, comprising, a) obtaining a nucleic acid sample from the subjectsuspected of having HSP or being at risk of developing HSP; and b)determining the presence or absence of a sequence defect in a sequenceencoding a polypeptide comprising the sequence of SEQ ID NO: 19 in thenucleic acid sample obtained from the subject, wherein the defectcomprises a mutation in the gene coding sequence, wherein the genecoding sequence is as set forth in SEQ ID NO: 18 the mutation beingselected from the group consisting of a substitution of a guanine atposition 2205 with a thymine, a substitution of a guanine at position2186 with a cytosine, and a substitution of an adenine at position 1740with a guanine, whereby the detection of the defect is indicative thatthe subject has or is at risk of developing SPG8-associated HSP.
 27. Themethod of claim 26 wherein the determining the presence or absence of asequence defect in the nucleic acid sample includes an amplificationstep.
 28. A method of detecting the presence or absence of a nucleotidesequence mutation in a strumpellin gene coding sequence, said methodcomprising the steps of: a) obtaining and analyzing a nucleic acid testsample containing all, or a portion of, the strumpellin gene codingsequence; b) comparing the results of said analysis of said sample ofstep a) with the results of an analysis of a control nucleic acid samplecontaining a wildtype strumpellin gene coding sequence, wherein thewildtype strumpellin gene encodes a polypeptide comprising the sequenceof SEQ ID NO: 19; and c) determining the presence or absence of at leastone mutation in the strumpellin gene coding sequence of the test sample,wherein the mutation results in a mutant polypeptide in which at leastone amino acid residue of SEQ ID NO: 19 is substituted with anotheramino acid residue, and wherein the at least one amino acid residue isselected from the group consisting of an asparagine residue at position471, a leucine residue at position 619 and a valine residue at position626.
 29. The method of claim 28 wherein the analyzing of the nucleicacid sample includes an amplification step.
 30. A method of detectingthe presence or absence of a nucleotide sequence mutation in astrumpellin gene coding sequence, said method comprising the steps of:a) obtaining and analyzing a nucleic acid test sample containing all, ora portion of, the strumpellin gene coding sequence; b) comparing theresults of said analysis of said sample of step a) with the results ofan analysis of a control nucleic acid sample containing a wildtypestrumpellin gene coding sequence, wherein the wildtype strumpellin geneencodes a polypeptide comprising the sequence of SEQ ID NO: 19; and c)determining the presence or absence of at least one mutation strumpellingene coding sequence of the test sample, wherein the mutation results ina mutant polypeptide in which amino acid residue 471 of SEQ ID NO: 19 issubstituted with an aspartate residue, or in which amino acid residue619 of SEQ ID NO: 19 is substituted with a phenylalanine residue or inwhich amino acid residue 626 of SEQ ID NO: 19 is substituted with aphenylalanine residue.
 31. The method of claim 30 wherein the analysisof the nucleic acid sample includes an amplification step.
 32. A methodof detecting the presence or absence of a nucleotide sequence mutationin a strumpellin gene coding sequence, said method comprising the stepsof: a) obtaining and analyzing a nucleic acid test sample containingall, or a portion of, the coding sequence; b) comparing the results ofsaid analysis of said sample of step a) with the results of an analysisof a control nucleic acid sample containing a wildtype strumpellin genecoding sequence, wherein the wildtype strumpellin gene encodes apolypeptide comprising the sequence of SEQ ID NO: 19; and c) determiningthe presence or absence of at least one mutation in the strumpellin genecoding sequence of the test sample, wherein the gene coding sequencecomprises SEQ ID NO: 18 and the mutation is selected from the groupconsisting of a substitution of a guanine at position 2205 with anothernucleotide, a substitution of a guanine at position 2186 with anothernucleotide, and a substitution of an adenine at position 1740 withanother nucleotide.
 33. The method of claim 32 wherein the analysis ofthe nucleic acid sample includes an amplification step.
 34. A method ofdetecting the presence or absence of a nucleotide sequence mutation in astrumpellin gene coding sequence, said method comprising the steps of:a) obtaining and analyzing a nucleic acid test sample containing all, ora portion of, the gene; b) comparing the results of said analysis ofsaid sample of step a) with the results of an analysis of a controlnucleic acid sample containing a wildtype strumpellin gene codingsequence, wherein the wildtype strumpellin gene encodes a polypeptidecomprising the sequence of SEQ ID NO: 19; and c) determining thepresence or absence of at least one mutation in the strumpellin genecoding sequence of the test sample, wherein the gene comprises SEQ IDNO: 18 and the mutation is selected from the group consisting of asubstitution of a guanine at position 2205 with a thymine, asubstitution of a guanine at position 2186 with a cytosine, and asubstitution of an adenine at position 1740 with a guanine.
 35. Themethod of claim 34 wherein the analysis of the nucleic acid sampleincludes an amplification step.
 36. A method of detecting the presenceor absence of a nucleotide sequence mutation in a strumpellin genecoding sequence, said method comprising the steps of: a) obtaining andanalyzing a nucleic acid test sample containing all, or a portion of,the coding sequence; b) comparing the results of said analysis of saidsample of step a) with the results of an analysis of a control nucleicacid sample containing a wildtype strumpellin gene coding sequence,wherein the wildtype strumpellin gene coding sequence comprises thesequence of SEQ ID NO: 18; and c) determining the presence or absence ofat least one mutation in the strumpellin gene coding sequence of thetest sample, wherein the mutation is selected from the group consistingof a substitution of a guanine at position 2205 with another nucleotide,a substitution of a guanine at position 2186 with another nucleotide,and a substitution of an adenine at position 1740 with anothernucleotide.
 37. The method of claim 36 wherein the analysis of thenucleic acid sample includes an amplification step.
 38. A method ofdetecting the presence or absence of a nucleotide sequence mutation in astrumpellin gene coding sequence, said method comprising the steps of:a) obtaining and analyzing a nucleic acid test sample containing all, ora portion of, the gene; b) comparing the results of said analysis ofsaid sample of step a) with the results of an analysis of a controlnucleic acid sample containing a wildtype strumpellin gene codingsequence, wherein the wildtype strumpellin gene coding sequencecomprises the sequence of SEQ ID NO: 18; and c) determining the presenceor absence of at least one mutation in the strumpellin gene codingsequence of the test sample, wherein the mutation is selected from thegroup consisting of a substitution of a guanine at position 2205 with athymine, a substitution of a guanine at position 2186 with a cytosine,and a substitution of an adenine at position 1740 with a guanine. 39.The method of claim 38 wherein the analysis of the nucleic acid sampleincludes an amplification step.
 40. A method for diagnosingSPG8-associated hereditary spastic paraplegia (HSP) in a human subject,comprising a) obtaining a nucleic acid sample from a subject who hasbeen pre-diagnosed as a likely candidate for developing HSP; b)determining the presence or absence of a nucleotide sequence defect inall, or a portion of, a strumpellin gene coding sequence contained inthe sample, wherein the strumpellin gene encodes a polypeptidecomprising all, or a portion of, SEQ ID NO: 19, and the sequence defectcomprises a mutation in the gene resulting in a mutant polypeptide inwhich at least one amino acid residue of SEQ ID NO: 19 is substitutedwith another amino acid residue, and wherein the at least one amino acidresidue is selected from the group consisting of an asparagine residueat position 471, a leucine residue at position 619 and a valine residueat position 626, wherein the presence of one, or more nucleotidesequence defects in the gene is indicative of SPG8-associated HSP in thesubject.
 41. The method of claim 40 wherein the sequence defectcomprises a mutation in the gene resulting in a mutant polypeptide inwhich amino acid residue 471 of SEQ ID NO: 19 is substituted with anaspartate residue, or in which amino acid residue 619 of SEQ ID NO: 19is substituted with a phenylalanine residue or in which amino acidresidue 626 of SEQ ID NO: 19 is substituted with a phenylalanineresidue.
 42. The method of claim 40, wherein the defect comprises amutation in the gene, wherein the gene coding sequence as set forth inSEQ ID NO: 18, the mutation being selected from the group consisting ofa substitution of a guanine at position 2205 with another nucleotide, asubstitution of a guanine at position 2186 with another nucleotide, anda substitution of an adenine at position 1740 with another nucleotide.43. The method of claim 40, wherein the defect comprises a mutation inthe gene, wherein the gene is as set forth in SEQ ID NO: 18, themutation being selected from the group consisting of a substitution of aguanine at position 2205 with a thymine, a substitution of a guanine atposition 2186 with a cytosine, and a substitution of an adenine atposition 1740 with a guanine.
 44. The method of claim 40 wherein thenucleic acid sample is amplified prior to determining the nucleotidesequence of the strumpellin gene coding sequence in the sample.
 45. Themethod of claim 1 wherein the nucleic acid sample is amplified prior todetermining the nucleotide sequence of the strumpellin gene codingsequence in the sample.
 46. The method of claim 1 wherein determiningthe presence or absence of a sequence defect comprises one or moremethods selected from the group consisting of: sequence analysis;fragment polymorphism assays; hybridization assays and computer baseddata analysis.
 47. The method of claim 16 wherein the determination ofthe presence or absence of a sequence defect includes analysis selectedfrom the group consisting of: sequence analysis; fragment polymorphismassays; hybridization assays and computer based data analysis.
 48. Themethod of claim 20 wherein the determination of the presence or absenceof a sequence defect includes analysis selected from the groupconsisting of: sequence analysis; fragment polymorphism assays;hybridization assays and computer based data analysis.
 49. The method ofclaim 22 wherein the determination of the presence or absence of asequence defect includes analysis selected from the group consisting of:sequence analysis; fragment polymorphism assays; hybridization assaysand computer based data analysis.
 50. The method of claim 24 wherein thedetermination of the presence or absence of a sequence defect includesanalysis selected from the group consisting of: sequence analysis;fragment polymorphism assays; hybridization assays and computer baseddata analysis.
 51. The method of claim 26 wherein the determination ofthe presence or absence of a sequence defect includes analysis selectedfrom the group consisting of: sequence analysis; fragment polymorphismassays; hybridization assays and computer based data analysis.
 52. Themethod of claim 28 wherein the determination of the presence or absenceof a mutation includes analysis selected from the group consisting of:sequence analysis; fragment polymorphism assays; hybridization assaysand computer based data analysis.
 53. The method of claim 30 wherein thedetermination of the presence or absence of a mutation includes analysisselected from the group consisting of: sequence analysis; fragmentpolymorphism assays; hybridization assays and computer based dataanalysis.
 54. The method of claim 32 wherein the determination of thepresence or absence of a mutation includes analysis selected from thegroup consisting of: sequence analysis; fragment polymorphism assays;hybridization assays and computer based data analysis.
 55. The method ofclaim 34 wherein the determination of the presence or absence of amutation includes analysis selected from the group consisting of:sequence analysis; fragment polymorphism assays; hybridization assaysand computer based data analysis.
 56. The method of claim 36 wherein thedetermination of the presence or absence of a mutation includes analysisselected from the group consisting of: sequence analysis; fragmentpolymorphism assays; hybridization assays and computer based dataanalysis.
 57. The method of claim 38 wherein the determination of thepresence or absence of a mutation includes analysis selected from thegroup consisting of: sequence analysis; fragment polymorphism assays;hybridization assays and computer based data analysis.
 58. The method ofclaim 40 wherein the determination of the presence or absence of asequence defect includes analysis selected from the group consist of:sequence analysis; fragment polymorphism assays; hybridization assaysand computer based data analysis.